Nanoparticles

ABSTRACT

Materials and methods for studying and modulating the interaction of carbohydrate-containing moieties with other species are described, in particular, small particles, e.g. clusters of metal or semiconductor atoms, which can be employed as a substrate for immobilising a plurality of ligands comprising carbohydrate groups. These “nanoparticles” can then be used to study carbohydrate mediated interactions, e.g. with other carbohydrates or proteins, and as therapeutics and diagnostic reagents.

This application is a division of U.S. Ser. No. 12/040,594, filed Feb.29, 2008, issuing, which is a continuation of U.S. application Ser. No.10/399,284, filed Oct. 29, 2003, now U.S. Pat. No. 7,364,919, which isthe U.S. national phase, pursuant to 35 U.S.C. §371, of PCTinternational application Ser. No. PCT/GB2001/04633, filed Oct. 16,2001, designating the United States and published in English on Apr. 25,2002 as publication WO 02/32404 A2, which claims priority to GreatBritain application Ser. No. 0025414.4, filed Oct. 16, 2000. The entirecontents of the aforementioned patent applications are incorporatedherein by this reference.

FIELD OF THE INVENTION

The present invention relates to nanoparticles, and in particular tonanoparticles having immobilised ligands comprising carbohydrate groupsand their use in studying the interaction of these ligands with otherspecies. The present invention further relates to applications of thenanoparticles, for example for screening, diagnosis and therapy.

BACKGROUND OF THE INVENTION

There are three major classes of biopolymers, nucleic acid, proteins andcarbohydrates. Protein and nucleic acid structure and interactions havebeen extensively studied in the art and the template-driven nature ofprotein and nucleic acid synthesis and the fact that these polymers arelinear has meant that the techniques for their production and study havenow been largely automated.

However, carbohydrates and their interactions with other species arealso extremely important biologically and have not been the subject ofconcerted study. The difficulty in studying carbohydrates and theirinteractions arises in view of the diversity of carbohydrate linkagesand because there are no techniques analogous to cloning to amplify andmodify carbohydrates. On the contrary, the complex multistep way inwhich carbohydrates are assembled in cells means that carbohydrates andassociated glycoconjugates such as glycoproteins and glycolipids arecharacterised by a high degree of variability and are not trivial tosynthesise or study. In addition, carbohydrate mediated interactionstend to be weak and polyvalent and are correspondingly difficult todetect. Thus, there are no satisfactory tools for doing this in the art.

However, despite these characteristics, carbohydrate mediatedinteractions are important biologically. The surfaces of most types ofcells are covered with a dense coating of glycoconjugates given rise tothe so-called glycocalyx. It is believed that the glycocalyx isresponsible for the repulsive forces which prevent non-specific adhesionof cells. However, in some cell configurations the repulsive barrierwill be counterbalanced by the formation of cell-cell contacts throughattractive forces.^([1]) There is now evidence that beside thewell-known carbohydrate-protein interactions,^([2]) cells use attractiveinteractions between surface carbohydrates as a novel mechanism for celladhesion and recognition.^([3]) A characteristic feature of theseinteractions is its low affinity that is compensated by a polyvalentpresentation of ligands and receptors at the cell surfaces.^([4])

Investigations into polyvalent carbohydrate-protein interactions havebeen approached using different multivalent carbohydrate modelsystems.^([5]) Examples of prior art approaches include the use of twodimensional arrays of glycoconjugates on gold surfaces ^([6a]), the useof liposomes to display carbohydrates, dendrimer technology, and the useof polymers to provide linear and spherical carbohydrate arrays^([5a,b]). However, the problems of studying interactions involvingcarbohydrates are far from solved and there is a continuing need in theart for new methods and tools for doing this.

SUMMARY OF THE INVENTION

Broadly, the present invention provides materials and methods forstudying and modulating the interaction of carbohydrate-containingmoieties with other species. In particular, the present inventionprovides small particles, e.g. clusters of metal or semiconductor atoms,which can be employed as a substrate for immobilising a plurality ofligands, the ligands comprising carbohydrate groups. These‘nanoparticles’ can then be used to study carbohydrate mediatedinteractions, e.g. with other carbohydrates or proteins, and astherapeutics and diagnostic reagents. Thus, the present inventionprovides a way of providing a spherical array of the ligand immobilisedon a detectable particle. In some embodiments, the particles have thefurther advantage that they are soluble, e.g. in water and a range oforganic solvents, and can be used in a variety of homogeneousapplication formats.

Accordingly, in a first aspect, the present invention provides aparticle comprising a core, such as a metallic core, linked to aplurality of ligands, wherein the ligands comprise a carbohydrate group.The ligands may comprise the carbohydrate groups alone or in combinationwith peptides, protein domains, nucleic acid segments or fluorescentgroups.

In a further aspect, the present invention provides compositionscomprising populations of one or more of the above defined particles. Insome embodiments, the populations of nanoparticles may have differentdensities of the same or different ligands attached to the core.

In a further aspect, the present invention provides the above definedparticles for use in a method of medical treatment.

In a further aspect, the present invention provides the use of the abovedefined particles for the preparation of a medicament for the treatmentof a condition ameliorated by the administration of the ligand. By wayof example, this may occur as the ligand blocks a carbohydrate mediatedinteraction that would otherwise tend to lead to a pathology.

In this embodiment, the present invention has advantages over prior artapproaches for treating conditions involving carbohydrate mediatedinteractions. As described above, typically the interactions arepolyvalent whereas the agent used to treat the interactions are oftenonly capable of modulating one or a few of the these interactions. Thishas the result that it is difficult to deliver an agent to the site ofthe interaction which is capable of reliably modulating the interactionfor the desired therapeutic effect. In contrast to this problem, thepresent invention provides agents having a plurality of ligands formodulating the carbohydrate mediated interactions, potentiallyovercoming the difficulty in modulating the polyvalent interactions.

In preferred embodiments, the mean diameter of the core, preferably themetallic core, is between 0.5 and 100 nm, more preferably between 1 and50 nm, and still more preferably between 1 and 20 nm. The mean diametercan be measured using techniques well known in the art such astransmission electron microscopy.

The core material can be a metal or semiconductor and may be formed ofmore than one type of atom. Preferably, the core material is a metalselected from Au, Ag or Cu. Nanoparticles cores formed from alloys havealso been reported, including Au/Ag, Au/Cu, Au/Ag/Cu, Au/Pt, Au/Pd andAu/Ag/Cu/Pd, and may be used in the present invention. Preferred corematerials are Au and Ag, with the most preferred material being Au.Where gold clusters are used, they will preferably have between about100 and 500 gold atoms to provide core diameters in the nanometer range.Other particularly useful core materials are or are doped with one ormore atoms that are NMR active, allowing the nanoparticles to bedetected using NMR, both in vitro and in vivo. Examples of NMR activeatoms include gadolinium and europium.

Nanoparticle cores comprising semiconductor atoms can be detected asnanometer scale semiconductor crystals are capable of acting as quantumdots, that is they can absorb light thereby exciting electrons in thematerials to higher energy levels, subsequently releasing photons oflight at frequencies characteristic of the material. An example of asemiconductor core material is cadmium selenide.

The nanoparticles and the results of their interactions can be detectedusing a number of techniques well known in the art. These can range fromdetecting the aggregation that results when the nanoparticles bind toanother species, e.g. by simple visual inspection or by using lightscattering (transmittance of a solution containing the nanoparticles),to using sophisticated techniques such as transmission electronmicroscopy (TEM) or atomic force microscopy (AFM) to visualise thenanoparticles. A further method of detecting metal particles is toemploy plasmon resonance, that is the excitation of electrons at thesurface of a metal, usually caused by optical radiation. The phenomenonof surface plasmon resonance (SPR) exists at the interface of a metal(such as Ag or Au) and a dielectric material such as air or water. Aschanges in SPR occur as analytes bind to the ligand immobilised on thesurface of a nanoparticle changing the refractive index of theinterface. A further advantage of SPR is that it can be used to monitorreal time interactions. As mentioned above, if the nanoparticlesincludes or is doped with atoms which are NMR active then this techniquecan be used to detect the particles, both in vitro or in vivo, usingtechniques well known in the art. Nanoparticles can also be detected asdescribed in ^([18]), using a system based on quantitative signalamplification using the nanoparticle-promoted reduction of silver (I)and using a flatbed scanner as a reader. Fluorescence spectroscopy canbe used if the nanoparticles include ligands combining carbohydrategroups and fluorescent probes. Also, isotopic labelling of thecarbohydrate can be used to facilitate their detection.

The ligand linked to the core comprises one or more carbohydrate(saccharide) groups, e.g. comprising a polysaccharide, anoligosaccharide or a single saccharide group. The ligand may be also bea glycanoconjugate such as a glycolipid or a glycoprotein. In additionto the carbohydrate group, the ligand may additionally comprises one ormore of a peptide group, a protein domain, a nucleic acid molecule (e.g.a DNA segment) and/or a fluorescent probe.

The particles may have more than one species of ligand immobilisedthereon, e.g. 2, 3, 4, 5, 10, 20 or 100 different ligands. Alternativelyor additionally a plurality of different types of particles can beemployed together.

In preferred embodiments, the mean number of ligands linked to anindividual metallic core of the particle is at least 20 ligands, morepreferably at least 50 ligands, and most preferably at least 100ligands. Preferred densities of ligands are in the ranges of 70-100ligands per 200 gold atoms as determined by elemental analysis.

Preferably, the ligands are attached covalently to the core of theparticles. Protocols for carrying this out are known in the art,although the work described herein is the first report of the reactionsbeing used to covalently bond carbohydrate ligands to the core of theparticle. This may be carried out by reacting ligands with reductive endgroups with gold under reducing conditions. A preferred method ofproducing the particles employs thiol derivatised carbohydrate moietiesto couple the ligands to particles. Thus, in one aspect, the presentinvention provides a method of preparing the above defined particles,the method comprising:

synthesizing a sulphide derivative of the ligand;

reacting the sulphide derivatised ligand and tetrachloroauric acid inthe presence of reducing agent to produce the particles.

In a preferred embodiment, the ligand is derivatised as a protecteddisulphide. Conveniently, the disulphide protected ligand in methanolcan be added to an aqueous solution of tetrachloroauric acid. Apreferred reducing agent is sodium borohydride. Other preferred featuresof the method are described in the examples below.

The present invention provides a way of presenting a spherical array ofcarbohydrate-containing ligands having advantages over other types ofarray proposed in the prior art. In particular, the nanoparticles aresoluble in most organic solvents and especially water. This can be usedin their purification and importantly means that they can be used insolution as macroarrays for presenting the ligand immobilised on thesurface of the particle. The fact that the nanoparticles are soluble hasthe advantage of presenting the carbohydrates in a natural conformation.For therapeutic applications, the nanoparticles are non-toxic, solubleand excreted in the urine.

A range of different carbohydrate mediated interactions are known in theart and could be studied or modulated using the nanoparticles disclosedherein. These include leukocyte-endothelial cell adhesion,carbohydrate-antibody interactions, carbohydrate-protein bacterial andviral infection, immunological recognition of tumour cells, tumourcells-endothelial cells (e.g. to study metastasis) and foreign tissueand cell recognition. The following examples of application for thenanoparticles are provided by way of illustration and not limitation tosupport the wide applicability of the technologies described herein.

In general, it has been a difficult problem in the art to detect ormodulate carbohydrate-mediated interactions since the binding ofcarbohydrates to other species such as proteins or other carbohydratesis very weak and tends to be polyvalent. Thus, for detection the bindingis weak and for modulating interaction, monovalent agents have only hada limited success in disrupting polyvalent carbohydrate basedinteractions.

In embodiments of the invention relating to carbohydrate-carbohydrateinteractions, two types of interaction can be identified. In homophilicinteractions, identical carbohydrates interact with one another andcould be detected by steadily increasing the concentration of particleshaving a single species of ligands immobilised on their surface untilaggregation occurs. This may be detected by light scattering orelectronic effects. Heterophilic interactions can be detected by mixingtogether two or more different nanoparticles and determining theaggregation state of the particles.

Thus, the present invention provides a versatile platform for studyingand modulating carbohydrate-mediated interactions. For example, theparticles could be used to detect anti-carbohydrate antibodies,detecting the binding of antibody to the ligands on the particle vialight scattering to pick up aggregation of the particles, or electricfield effects, such as surface plasmon resonance, which would bemodified when the metal atoms in the particles cluster together.

In one example of this aspect of the invention, the nanoparticles can beemployed to type blood groups, as commonly carried out in medicine tomatch compatible donors and recipients for blood transfusion. Bloodgroups arise as common gut bacteria bear carbohydrate antigens which aresimilar or identical to blood group antigens present on the surface ofred blood cells, and these bacterial antigens stimulate the productionof antibodies in individuals who do not bear the corresponding antigenon their own red blood cells. Thus, sera from an individual is testedfor antibodies that agglutinate the red blood cells of the donor andvice versa in a cross-match test to detect the potentially harmfulantibodies in the recipient. At present, blood typing is carried outusing these agglutination tests which are inconvenient and not readilysusceptible to automation or high throughput.

The blood group antigens are carbohydrates, e.g. for the common antigen:

Type O R-GlcNAc-Gal(Fuc) Type A R-GlcNAc-Gal(Fuc)-GalNAc Type BR-GlcNAc-Gal(Fuc)-Gal Type AB Type A and Type B antigens

Therefore, populations of nanoparticles can be made having blood groupantigens immobilised on their surface. Thus, if a sample contained seracapable of binding to the blood group antigen, then adding thenanoparticle to a sample from a patient would allow the blood type ofdonors and recipients to be determined.

Another application of the nanoparticles is to modulate inflammation. Inparticular, members of the selectin family participate in the initialattachment of white blood cells (leukocytes) to endothelial cells duringthe process of leukocyte recruitment to inflammed tissues. L-selectin isexpressed on leukocytes, P-selectin on platelets and E-selectin onendothelial cells. E-selectin and P-selectin are induced on endothelialcells in response to pro-inflammatory cytokines and bind to ELAMreceptors on the surface of endothelial cells. L-selectin isconstitutively expressed on circulating leukocytes and binds toglycoproteins uniquely expressed on the activated endothelium. Thus, allof these interactions could be employed as therapeutic targets formodulating inflammation, and in particular reduce aberrant inflammation.

Prior art approaches to employing selectins as therapeutic targets havebeen based on the fact that the selectins share a common calciumdependent lectin domain which can be targeted by carbohydrate basedligands. Prior art screening has found that all three selectins bind tothe sialylated and fucosylated tetrasaccharide sialyl Lewis X (sLe^(x))and that this molecule and analogues thereof can bind to selectin,albeit weakly. The prior art approaches suffer from the problem that theinteraction of sLe^(x) and selectin is weak and the interaction of thecells expressing the selectin is polyvalent. Accordingly, in one of itsaspects, the present invention proposes a treatment of inflammationusing nanoparticles having one or more selectin ligands immobilisedthereon. A discussion of selectin mediated inflammation and compoundsthat can be used to modulate the interaction is provided in^([5b]).

In a further aspect, nanoparticles in which the carbohydrate(saccharide) group is an antigen can be administered as a vaccine, e.g.ballistically, using a delivery gun to accelerate their transdermalpassage through the outer layer of the epidermis. The nanoparticles canthen be taken up, e.g. by dendritic cells, which mature as they migratethrough the lymphatic system, resulting in modulation of the immuneresponse and vaccination against the saccharide antigen, as describedin^([19]).

In a further application, it is known that cell surface carbohydratesact as ligands for viral or bacterial receptors (called adhesins) andthat binding of the carbohydrates to the receptors is an event requiredduring infection. Synthetic carbohydrates, e.g. glycoconjugates, thatare capable of modulating these interactions can be immobilised in thenanoparticles of the invention and used as reagents to study theseinteractions and as therapeutics to prevent viral or bacterialinfection.

One example of a carbohydrate ligand mediating bacterial infection isHelicobacter pylori which causes chronic active gastritis, gastric andduodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoidtissue lymphoma in humans. As the cell specific attachment of H. pylorican occur via multiple carbohydrates including Lewis b antigen,sialylated oligosaccharides and sulphated mucin glycoproteins,nanoparticles capable of modulating (i.e. blocking) the differentadhesin interactions could be used as treatments of the aboveconditions.

Examples of viral infections mediated by carbohydrates include theinfluenza virus which infects cells via the multivalent binding ofhemaglutinin molecules on the viral envelope to sialic acid terminatedhost glycoconjugates. Thus, by infection may be inhibited by disruptingthis event.

HIV-1 also infects cells by recognising cell surface carbohydratestructures and the glycolipid galactosylceramide (GalCer) has beenidentified as a ligand for the HIV-1 receptor gp120. Thus, GalCer oranalogues thereof could be immobilised on the surface of nanoparticlesand used to inhibit the interaction of cellular GalCer and HIV-1.

In a further application, the present invention may be useful in themodulation of immune response, e.g. following transplantation. As theimmunological recognition of tissue begins with carbohydrate mediatedinteractions between surface carbohydrates present on transplantedtissue and the components of the host's immune system such asantibodies, so this can be targeted to ameliorate immune reactions thatresult from this interaction. By way of example the carbohydrateGalα1-3Galβ1-4GlnAc (the ‘αGal’ epitope) has been implicated as animportant antigenic epitope involved in the rejection of transplantedtissue. Thus, modulation of the interaction of the αGal epitope and theimmune system may be a therapeutic target for the nanoparticlesdescribed herein.

An alternative approach may be useful in the treatment of cancer as manytumour associated antigens or tumour autocrine factors are carbohydratebased. In this event, the nanoparticles could be provided as vaccinesprime the immune system to produce antibodies which are capable ofattacking tumour cells presenting the carbohydrates on their surface. Inthis regard, it is known that many tumour cells possess aberrantglycosylation patterns which may enable the immune response stimulatedby nanoparticles to be directed specifically to tumour cells as opposedto normal, healthy cells. The nanoparticles can also be used to inhibitmetastatis in cancer, e.g. through the migration of tumour cells throughthe endothelial cells.

In a further aspect, the nanoparticles can be used as carriers to raiseantibodies capable of specifically binding the ligand. This isparticularly advantageous as it can be a challenging problem in the artto raise antibodies against carbohydrates-containing moieties as theyare often small and do not cause strong immune responses.

In a further aspect, the present invention provides a method ofdetermining whether a carbohydrate mediated interactions occurs, themethod comprising contacting one or more species of nanoparticles with acandidate binding partner and determining whether binding takes place.

In a further aspect, the present invention provides a method ofscreening for substances capable of binding to a ligand comprising acarbohydrate group, the method comprising:

-   -   contacting particles comprising a metallic core linked to a        plurality of the ligands with one or more candidate compounds;        and    -   detecting whether the candidate compounds binds to the ligand.

In a further aspect, the present invention provides a method ofdetermining the presence in a sample of a substance capable of bindingto a ligand comprising a carbohydrate ligand, the method comprisingcontacting the sample with nanoparticles linked to the ligand anddetermining whether binding takes place. The method may be used todetermine the presence or amount of one or more analytes in a sample,e.g. for use in assisting the diagnosis of a disease state associatedwith the presence of the analyte.

In a further embodiment, the nanoparticles can be employed to study ordetect carbohydrate mediated interactions in conjunction with speciesimmobilised on solid surfaces, for example ligands immobilised on goldsurfaces as described in ^([6a]). These species might be othercarbohydrates, candidate binding partners or analytes.

In a further aspect, carbohydrates can be attached to nanocrystals ofcadmium selenide to provide quantum dots, which can then be guided tothe required cellular structure by nanoparticles. As discussed in^([20]), quantum dots have potential uses in biological imaging, in bothelectronic and optical devices, quantum computers and the screening ofcandidate drugs.

Embodiments of the present invention will now be described by way ofexample and not limitation with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows schematically the method used to synthesise thenanoparticles.

FIG. 2 shows transmission electron micrographs and core sizedistribution histograms (insets) of the lacto 2-Au (top) and the Le^(x)3-Au (bottom) glyconanoparticles.

FIG. 3 shows ¹H NMR spectra of: (A) 2-Au in D₂O (a); 2 in D₂O (b) and 2in CD₃OD (c) and (B) 3-Au in D₂O (a); 3 in D₂O (b) and 3 in 70%CD₃OD/D₂O (c).

DETAILED DESCRIPTION

Pharmaceutical Compositions

The nanoparticles described herein or their derivatives can beformulated in pharmaceutical compositions, and administered to patientsin a variety of forms, in particular to treat conditions ameliorated bythe administration of the ligand. By way of example, this may occur asthe ligand blocks a carbohydrate mediated interaction that wouldotherwise tend to lead to a pathology. Thus, the nanoparticles may beused as medicament for modulating leukocyte-endothelial cell adhesion,carbohydrate-antibody interactions, carbohydrate-protein bacterial andviral infection, immunological recognition of tumour cells, theinhibition of metastatis and foreign tissue and cell recognition.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant or an inert diluent. Liquidpharmaceutical compositions generally include a liquid carrier such aswater, petroleum, animal or vegetable oils, mineral oil or syntheticoil. Physiological saline solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol may be included. Suchcompositions and preparations generally contain at least 0.1 wt % of thecompound.

Parenteral administration includes administration by the followingroutes: intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraocular, transepithelial, intraperitoneal and topical (includingdermal, ocular, rectal, nasal, inhalation and aerosol), and rectalsystemic routes. For intravenous, cutaneous or subcutaneous injection,or injection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, solutions of the compounds or a derivative thereof,e.g. in physiological saline, a dispersion prepared with glycerol,liquid polyethylene glycol or oils.

In addition to one or more of the compounds, optionally in combinationwith other active ingredient, the compositions can comprise one or moreof a pharmaceutically acceptable excipient, carrier, buffer, stabiliser,isotonicizing agent, preservative or anti-oxidant or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material maydepend on the route of administration, e.g. orally or parenterally.

Liquid pharmaceutical compositions are typically formulated to have a pHbetween about 3.0 and 9.0, more preferably between about 4.5 and 8.5 andstill more preferably between about 5.0 and 8.0. The pH of a compositioncan be maintained by the use of a buffer such as acetate, citrate,phosphate, succinate, Tris or histidine, typically employed in the rangefrom about 1 mM to 50 mM. The pH of compositions can otherwise beadjusted by using physiologically acceptable acids or bases.

Preservatives are generally included in pharmaceutical compositions toretard microbial growth, extending the shelf life of the compositionsand allowing multiple use packaging. Examples of preservatives includephenol, meta-cresol, benzyl alcohol, para-hydroxybenzoic acid and itsesters, methyl paraben, propyl paraben, benzalconium chloride andbenzethonium chloride. Preservatives are typically employed in the rangeof about 0.1 to 1.0% (w/v).

Preferably, the pharmaceutically compositions are given to an individualin a “prophylactically effective amount” or a “therapeutically effectiveamount” (as the case may be, although prophylaxis may be consideredtherapy), this being sufficient to show benefit to the individual.Typically, this will be to cause a therapeutically useful activityproviding benefit to the individual. The actual amount of the compoundsadministered, and rate and time-course of administration, will depend onthe nature and severity of the condition being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed), 1980. By way of example, and thecompositions are preferably administered to patients in dosages ofbetween about 0.01 and 100 mg of active compound per kg of body weight,and more preferably between about 0.5 and 10 mg/kg of body weight.

Antibodies

The nanoparticles may be used as carriers for raising antibody responsesagainst the carbohydrate containing ligands linked to the coreparticles. These antibodies can be modified using techniques which arestandard in the art. Antibodies similar to those exemplified for thefirst time here can also be produced using the teaching herein inconjunction with known methods. These methods of producing antibodiesinclude immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheepor monkey) with the nanoparticle(s). Antibodies may be obtained fromimmunised animals using any of a variety of techniques known in the art,and screened, preferably using binding of antibody to antigen ofinterest. Isolation of antibodies and/or antibody-producing cells froman animal may be accompanied by a step of sacrificing the animal.

As an alternative or supplement to immunising a mammal with ananoparticle, an antibody specific for the ligand and/or nanoparticlemay be obtained from a recombinantly produced library of expressedimmunoglobulin variable domains, e.g. using lambda bacteriophage orfilamentous bacteriophage which display functional immunoglobulinbinding domains on their surfaces; for instance see WO92/01047. Thelibrary may be naive, that is constructed from sequences obtained froman organism which has not been immunised with any of the nanoparticles,or may be one constructed using sequences obtained from an organismwhich has been exposed to the antigen of interest.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogenous population of antibodies, i.e.the individual antibodies comprising the population are identical apartfrom possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies can be produced by the method firstdescribed by Kohler and Milstein, Nature, 256:495, 1975 or may be madeby recombinant methods, see Cabilly et al, U.S. Pat. No. 4,816,567, orMage and Lamoyi in Monoclonal Antibody Production Techniques andApplications, pages 79-97, Marcel Dekker Inc, New York, 1987.

In the hybridoma method, a mouse or other appropriate host animal isimmunised with the antigen by subcutaneous, intraperitoneal, orintramuscular routes to elicit lymphocytes that produce or are capableof producing antibodies that will specifically bind to the nanoparticlesused for immunisation. Alternatively, lymphocytes may be immunised invitro. Lymphocytes then are fused with myeloma cells using a suitablefusing agent, such as polyethylene glycol, to form a hybridoma cell, seeGoding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986).

The hybridoma cells thus prepared can be seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody producingcells, and are sensitive to a medium such as HAT medium.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against thenanoparticles/ligands. Preferably, the binding specificity is determinedby enzyme-linked immunoabsorbance assay (ELISA). The monoclonalantibodies of the invention are those that specifically bind to thenanoparticles/ligands.

In a preferred embodiment of the invention, the monoclonal antibody willhave an affinity which is greater than micromolar or greater affinity(i.e. an affinity greater than 10⁻⁶ mol) as determined, for example, byScatchard analysis, see Munson & Pollard, Anal. Biochem., 107:220, 1980.

After hybridoma cells are identified that produce neutralisingantibodies of the desired specificity and affinity, the clones can besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include Dulbecco's ModifiedEagle's Medium or RPM1-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumours in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Nucleic acid encoding the monoclonal antibodies of the invention isreadily isolated and sequenced using procedures well known in the art,e.g. by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of murineantibodies. The hybridoma cells of the invention are a preferred sourceof nucleic acid encoding the antibodies or fragments thereof. Onceisolated, the nucleic acid is ligated into expression or cloningvectors, which are then transfected into host cells, which can becultured so that the monoclonal antibodies are produced in therecombinant host cell culture.

Hybridomas capable of producing antibody with desired bindingcharacteristics are within the scope of the present invention, as arehost cells containing nucleic acid encoding antibodies (includingantibody fragments) and capable of their expression. The invention alsoprovides methods of production of the antibodies including growing acell capable of producing the antibody under conditions in which theantibody is produced, and preferably secreted.

Antibodies according to the present invention may be modified in anumber of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus, the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope, here a carbohydrate ligand as definedherein.

Examples of antibody fragments, capable of binding an antigen or otherbinding partner, are the Fab fragment consisting of the VL, VH, Cl andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)₂ fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

A hybridoma producing a monoclonal antibody according to the presentinvention may be subject to genetic mutation or other changes. It willfurther be understood by those skilled in the art that a monoclonalantibody can be subjected to the techniques of recombinant DNAtechnology to produce other antibodies, humanised antibodies or chimericmolecules which retain the specificity of the original antibody. Suchtechniques may involve introducing DNA encoding the immunoglobulinvariable region, or the complementarity determining regions (CDRs), ofan antibody to the constant regions, or constant regions plus frameworkregions, of a different immunoglobulin. See, for instance, EP 0 184 187A, GB 2 188 638 A or EP 0 239 400 A. Cloning and expression of chimericantibodies are described in EP 0 120 694 A and EP 0 125 023 A.

EXPERIMENTAL

As strategy for tailoring polyvalent carbohydrate surfaces with globularshapes to investigate in solution carbohydrate-to-carbohydraterecognition, an approach was devised by which carbohydrates are linkedto gold nanoparticles^([7]). By way of example, the preparation,characterisation and preliminary interaction studies of sugarfunctionalized monolayer and water soluble gold nanoclusters isdisclosed below. Thiol derivatised neoglycoconjugates of two biologicalsignificant oligosaccharides, the lactose disaccharide(Galβ(1→4)Glcβ1-OR) 1 and 2 and of the trisaccharide Le^(x) antigen:(Galβ(1→4)[Fucα(1→3)]GlcNAcβ1-OR)  3have been prepared to attach them to gold nanoparticles.

The trisaccharide Le^(x) and the disaccharide lactose build up theglycosphingolipid (GSL) Le^(x) antigen:(Galβ1→4[Fuc1-3]GlcNAcβ1-3Galβ1-4Glcβ1-OCer)which have been proposed to mediate morula compaction and metastasis inhealthy and carcinoma mouse cells respectively, via a homotypiccarbohydrate-to-carbohydrate interaction.^([8])

The lacto and Le^(x) protected glyconanoparticles provide aglycocalyx-like surface with chemically well defined synthetic matrixand globular shape. Moreover, this approach opens the way to tailorglyconanoparticles containing a variety of carbohydrate ligands as wellas different surface density providing an under-control model forstructure-function studies and for investigating carbohydrateclustering^([9]) and orientation effects at a surface.^([10]) The lacto-and Le^(x) functionalised nanoparticles will be our polyvalent modelsystem to mimic GSL clustering in plasma membrane^([11]) and toinvestigate in solution the attractive and repulsive forces involved incell aggregation via carbohydrate-to-carbohydrate interactions. Previousstudies using synthetic receptors provided first solid evidence thatstabilising interactions between lipophilic carbohydrate surfaces existin water.^([12])

The synthesis of the disulfides 1, 2 and 3 was carried out byglycosidation of the conveniently protected lactose and Le^(x)derivative with 11-thioacetate-3,6,9-trioxa-undecanol (for 1) and11-thioacetate undecanol (for 2 and 3) using the trichloroacetimidatemethod, see FIG. 1.^([13]) Compounds 1, 2 and 3 were isolated asdisulfide forms, and in this form used for the formation of the goldprotected glyconanoparticles. The water soluble glyconanoparticles 1-Au,2-Au and 3-Au were obtained in methanol following the procedure of Brustet al for the synthesis of monolayer protected gold nanoclusters.^([7a])A series of gold protected nanoparticles, all of them soluble in organicsolvents, have recently been prepared for different purposes.^([14]) Thefacto-Au and Le^(x)-Au glyconanoparticles are water soluble, stable andcan be manipulated as a water soluble biological macromolecules. Theyhave been purified by dialysis and characterised by ¹H-NMR, UV andtransmission electron microscopy (TEM).

Synthesis of glyconanoparticles: A solution of disulphide 1,2, or 3(0.012M, 5.5 eq) in MeOH was added to a solution of tetrachloroauricacid (0.025M, 1 eq) in water. NaBH₄ (1M, 22 eq) in water was added insmall portions with rapid stirring. The black suspension that was formedwas stirred for additional 2 hours and the solvent was then removedunder vacuum. The crude of the reaction was washed with MeOH and wascentrifuged for 10 minutes. The methanol was removed and the process wasrepeated several times until the starting material was not detected byTLC. The glyco-nanoparticles are completely insoluble in MeOH but quitesoluble in water.

-   -   They were purified by dialysis: 50 mg of crude product was        dissolved in 10 mL of water (NANOpure). This solution was loaded        into 10 cm segments of cellulose ester dialysis membrane (SIGMA,        MWCO=12400) and placed in 4 L of water (NANOpure). The dark        glyconanoparticles solution was collected from the dialysis        segments and lyophilized. The products obtained were free of        salts and starting material (absence of signals due to        disulphide and Na⁺ in NMR).

Transmission Electron Microscopy (TEM) examination of the samples wascarried out with a Philips CM200 microscope working at 200 kV. A singledrop of a 0.1 mg/ml aqueous solution of the gold glyconanoparticles wasplaced onto a copper grid coated with a carbon film. The grid was leftto dry in air for several hours at room temperature. Particle sizedistribution of the Au clusters were evaluated from several micrographsusing an automatic image analyser. The number of particles selected forconsideration was around 400, which resulted in stable size distributionstatistic.

FIG. 2 shows TEM images and core size distribution histograms for the2-Au and 3-Au gold glyconanoparticles. The gold particles stabilizedwith the lactose show a narrower and more homogeneous particle sizedistribution than the particles stabilized with the Le^(x) conjugate. Amean diameter of 1.8 nm was found in both samples for the gold core ofthe functionalized nanoparticles. Such a mean particle size corresponds,according to previous work,^([15]) to an average number of gold atomsper particles of ca. 200 and 70 protecting alkanethiolateglycoconjugates. The aqueous solutions of the nanoparticles were stableduring months and no agglomeration was detected by TEM.

The presentation of the carbohydrate molecules at the nanoparticlessurface was then investigated. The molecular properties of theneoglycoconjugates 1, 2 and 3 suffer a differential change afterattaching them to the gold surface. For example, the lacto derivative 2,which is soluble in methanol and insoluble in water, givesglyconanoparticles 2-Au insoluble in methanol but with good solubilityin water. The Le^(x) derivative 3 is soluble in methanol and water, itsnanoparticle 3-Au, however, is insoluble in methanol and very soluble inwater. These differences in solubility can be used to purify theglyconanoparticles from the non-reacted disulfides by washing them withmethanol. However, the most significant fact in these changes is thatthey reveal the influence of clustering at the surface on thecarbohydrate presentation to the surrounding.

The ¹H-NMR spectra of the glyconanoparticles show clearly thesedifferences (FIG. 3). The spectra of the lacto-nanoparticles 1-Au and2-Au in D₂O differ strongly from those of the lacto-disulfides 1 and 2(FIG. 3A spectrum of 1 not shown) showing the line broadening of slowlyrotating macromolecules in solution. The signal of the methylenesclosest to the thiolate/Au interface completely disappears, as it occursin the alkanethiol monolayer-protected gold nanoclusters. In contrast,these differences are not founded in the case of the 3-Au nanoparticles.The ¹H-NMR spectra in D₂O of both 3 and 3-Au show similar broadening forall signals (FIG. 3B, a, b), indicating an intramolecular aggregationalready present in the Le^(x) disulfide 3. This self-interactionpersists even at highly diluted water solution and is abolished byaddition to the D₂O solution of 3 of increasing amounts of CD₃OD. Somewell-resolved signals appear in CD₃OD/D₂O (1:1) solution and in 70%CD₃OD/D₂O solution all signal are well-resolved in the spectrum (FIG.3B, c). The tendency of the Le^(x) disulfide 3 to self-assemble in watercannot exclusively be attributed to the hydrophobicity of the aliphaticchain, but rather to the specific partaking of the carbohydrate moietyin this aggregation, as point out the lack of aggregation in waterobserved in the ¹H-NMR of the lacto-disulfides 1 and 2 (FIG. 3A). Theself-aggregation ability will have consequences in the organisation andclustering of Le^(x)-containing GSLs, as claimed by some authors^([16])and contrary to the proposal of others that the carbohydrate head groupplays an insignificant role in formation of glycolipid-enrichedmicrodomains in the plasma membrane.^([17])

The steric crowding of the carbohydrate moiety at the nanoparticlesurfaces is also shown by the different behaviour of 1 and 2 and theircorresponding nanoclusters 1-Au and 2-Au with β-glycosidases. Theβ-galactosidase of E. coli processes 1 and 2 at a level comparable tolactose itself (5-10% relative to the specific activity of GONP), whilethe hydrolysis by the enzyme under the same conditions of 1-Au and 2-Aunanoparticles was barely detected (<3% relative to the enzymaticactivity with the free ligands 1 and 2).

These experiments demonstrate that it is possible to use nanoparticlesto produce tailored globular carbohydrate models mimicking GSL-clustersin plasma membrane, allowing for the first time investigations to becarried out in solution of a novel mechanism of cell adhesion viacarbohydrate-to-carbohydrate interactions. The glyconanoparticleapproach described herein provides a strategy to prepare, in a simpleway, a great variety of globular carbohydrate arrays that canadvantageously compete with other spherical (dendrimers, liposome) orlinear carbohydrate displays. The lacto- and Le^(x)-nanoparticles may beconsidered appropriate models to intervene in cell-cell adhesion andrecognition processes.

REFERENCES

The references mentioned herein are all expressly incorporated byreference.

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The invention claimed is:
 1. A nanoparticle comprising a core of metalatoms and/or semiconductor atoms covalently linked to at least onespecies of ligand, wherein the core of the nanoparticle is covalentlylinked to at least 20 ligands that each independently comprise acarbohydrate group.
 2. The nanoparticle of claim 1, wherein the core iscovalently linked to at least 50 ligands that each independentlycomprise a carbohydrate group.
 3. The nanoparticle of claim 1, whereinthe core of the nanoparticle has a mean diameter between 0.5 and 100nm.4. The nanoparticle of claim 1, wherein the metallic core comprises Au,Ag or Cu.
 5. The nanoparticle of claim 1, wherein the core comprises anatom which is NMR active.
 6. The nanoparticle of claim 1, wherein thecore includes an atom which is capable of detection using surfaceplasmon resonance.
 7. The nanoparticle of claim 1, wherein the ligandsfurther comprise a label.
 8. The nanoparticle of claim 1, wherein themetallic core is an alloy selected from Au/Ag, Au/Cu, Au/Ag/Cu, Au/Pt,Au/Pd or Au/Ag/Cu/Pd.
 9. The nanoparticle of claim 4, wherein metalliccore comprises between about 100 and 500 Au atoms.
 10. The nanoparticleof claim 1, wherein the ligands further comprise a peptide, a proteindomain, a nucleic acid segment or a fluorescent group.
 11. Thenanoparticle of claim 1, wherein ligand comprises a polysaccharide, anoligosaccharide or a monosaccharide group.
 12. The nanoparticle of claim1, wherein the ligand comprises a glycanoconjugate.
 13. The nanoparticleof claim 1, wherein the ligand is linked to the metallic core via asulphide group.
 14. The nanoparticle of claim 1, wherein thenanoparticle is water soluble.
 15. The nanoparticle of claim 1, whereinthe nanoparticle has more than one species of ligand immobilizedthereon.
 16. The nanoparticle of claim 15, wherein a further species ofligand comprises a peptide ligand, a protein domain ligand, a nucleicacid ligand or a fluorescent group ligand.
 17. The nanoparticle of claim1, wherein the nanoparticle further comprises a fluorescent label, anisotopic label, a NMR active atom, or a quantum dot.